To enable students to master the determination of urea nitrogen, this experiment is from the Mudanjiang Medical College undergraduate 5-year laboratory guide for laboratory tests.

Operation method

Experimental determination of serum urea nitrogen by diacetyl-oxime method

Principle

When heated in an acidic reaction environment, urea condenses with diacetyl to produce the pigmented proto-diazine, known as the Feorin reaction. Because diacetyl is unstable, it is usually produced by the reaction system in which the diacetyl-oxime interacts with a strong acid to produce diacetyl, which reacts with urea and synthesizes to produce the red diazine, whose shade of color is directly proportional to the amount of urea in the serum.

Move

I. Experimental reagents:

l. Alkaline reagent: in a triangular flask with distilled water about 100 ml, then add 44 ml of concentrated sulfuric acid and 85% H3PO466 ml cold to room temperature, add thiosemicarbazone 50 mg and cadmium sulphate 2 g to dissolve the distilled water diluted to 1 liter, placed in a brown bottle and put in the refrigerator to save, can be stable for half a year.

2. Diacetyl-oxime solution: weigh 20g of Diacetyl-oxime, add about 900ml of distilled water to dissolve, and then diluted to 1 liter of distilled water in a brown bottle, stored in the refrigerator can be kept for half a year.

3. Urea standard storage solution (100mmol/L): weigh dry pure urea (MW=60.06) 0.6g, dissolve in water and dilute to 100ml, add 0.1g sodium azide antiseptic, put in the refrigerator for six months.

4. Urea standard application solution (5mmol/L): take 5.0ml of storage solution and dilute it to 100ml with de-ammoniated distilled water.

II. Experimental operation:

III.Calculation:

Serum urea nitrogen mmol/L = (absorbance of measurement tube / absorbance of standard tube) × 5

Serum urea nitrogen mmol/L= urea mmol/L×28

Reference value: 2.86 - 8.2 mmol/L urea

Caveat

l. If serum or plasma urea nitrogen exceeds 400 mmol/L, the specimen must be diluted with saline before measurement, and the result is reported multiplied by the dilution factor.

2. micropipettes need to be calibrated, kept clean and dry, and the colorant added along the wall of the test tube after the sample has been added.

3， the reagent added thiosemicarbazone and cadmium sulfate, to enhance the intensity of color development and color stability, but there is still a mild discoloration phenomenon (＜5% per hour), heating color cooling, should be timely colorimetry.

4. Urea in urine can also be used for the determination of this method, due to the high content of urea in urine, the specimen should be distilled water for l: 50 times dilution, if the absorbance after color development is still more than the linear range of the method, it is also necessary to dilute the diluted urine and then re-diluted, re-determination.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/